IDENTIFICATION OF ISOLATED GENOMIC NUCLEOTIDE FRAGMENTS FROM THE p15 REGION OF CHROMOSOME 11 ENCODING HUMAN ACHAETE-SCUTE HOMOLOG 2 (HASH2) AND VARIANTS THEREOF

ABSTRACT

Provided herein are isolated genomic polynucleotide fragments from the p15 arm of chromosome 11 encoding HASH2 and methods of use.

PRIORITY CLAIM

This application is a divisional of application Ser. No. 13/239,243,filed Sep. 21, 2011, which is a divisional of application Ser. No.09/999,121 filed Oct. 31, 2001. The contents application Ser. No.13/239,243 and application Ser. No. 09/999,121 are incorporated hereinby reference. Application Ser. No. 09/999,121 also claims priority under35 U.S.C. 119(e) from provisional application Ser. No. 60/244,705, filedOct. 31, 2000, the contents of which are incorporated herein byreference.

FIELD OF THE INVENTION

The invention is directed to isolated genomic polynucleotide fragmentslocated in the p15 region of chromosome 11.

BACKGROUND OF THE INVENTION

Chromosome 11 contains genes encoding, for example, KCNQ1, avoltage-gated potassium channel; IPL, a homolog of a mouseapoptosis-inducing entity; human achaete-scute homolog 2 (HASH2), humanSMS3, human tumor suppressing subtransferable candidate 6 (TSSC6), humanribosomal protein L26 (RIBO26), cluster of differentiation antigen 81(CD81) and tumor suppressing subtransferable candidate 4 (TSSC4). Humanachaete-scute homolog 2 (HASH2), human SMS3, human tumor suppressingsubtransferable candidate 6 (TSSC6), human ribosomal protein L26(RIBO26), cluster of differentiation antigen 81 (CD81) and tumorsuppressing subtransferable candidate 4 (TSSC4) are discussed in furtherdetail below. Genes for the latter six proteins are located in the p15region of chromosome 11, a region known to be associated with theBeckwith-Wiedemann Syndrome (Itoh et al. Am. J. Genet. 92, 111-6, 2000)and some childhood tumors.

Beckwith-Wiedemann Syndrome is characterized by pre and postnatalovergrowth up to 160% of normal birthweight, macroglossia, hypoglycemia,hemi-hypertrophy and childhood tumors, such as Wilm's tumor (Reik etal., 1998, Trends Genet. 13:330-334). This syndrome appears to beassociated with deregulation of imprinting. Imprinted genes are genesthat are predominantly expressed from one of the parental chromosomes.There appears to be two imprinted subdomains, since the imprinted genedomain of 11p15 contains at least two imprinted subdomains (Lee et al.,1999, Hum. Mol. Genet. 8:683-690). Mosaicism may also play some role inthe Beckwith-Wiedemann Syndrome phenotype and may explain the variablephenotypes in Beckwith-Wiedemann Syndrome patients (Itoh et al., 2000,Am. J. Med. Genet. 92:111-116).

Human Achaete-Scute Homolog 2 (HASH2)

HASH2 is a basic helix-loop-helix protein that serves as a criticaltranscription factor for the development of the trophectoderm. Micedeficient in the HASH2 homolog, MASH2, die 10 days postcoitum due toplacental failure (Guillemot et al., Nature 371, 333-6, 1994).

Human Tumor Suppressing Subtransferable Candidates 4 and 6 (TSSC4 andTSSC6)

Both TSSC 4 and TSSC6 are believed to function as tumor-suppressingproteins in that the genes are among the genes of a subchromosomalfragment that suppresses in vitro growth of the rhabdomyosarcoma cellline RD (Koi et al., Science 260, 361-4, 1993).

Human Ribosomal Protein L26 (RIBO26)

RIBO26 is one of the approximately 80 proteins that compose the humanribosome (Kenmochi, N. et al., Genome Res. 8, 509-23, 1998). It has beenfound in mice to be induced by LPS and IFN gamma but is down regulatedby TNF-alpha (Segade et al., 1996, Life Sci. 58:277-285).

Human Cluster of Differentiation Antigen 81 (CD81)

CD81 (also called TAPA1) binds the E2 envelope protein of the humanhepatitis C virus and is believed to play a role in hepatitis Cinfection (Pileri et al., Science 282, 938-41, 1998). CD81 also appearsto play a role in T cell activation (Witherden et al., 2000, J. Immunol.165:1902-1909).

OBJECTS OF THE INVENTION

Although cDNAs encoding the above-disclosed proteins have been isolated,their precise locations and exon/intron/regulatory element organizationson chromosome 11 have not been determined. Furthermore, genomic DNAencoding these polypeptides have not been isolated. Noncoding sequencesplay a significant role in regulating the expression of polypeptides aswell as the processing of RNA encoding these polypeptides.

There is clearly a need for obtaining genomic polynucleotide sequencesencoding these polypeptides. Therefore, it is an object of the inventionto isolate such genomic polynucleotide sequences.

There is also a need to develop means for identifying mutations,duplications, translocations, polysomies and mosaicism associated withBeckwith-Wiedemann syndrome.

SUMMARY OF THE INVENTION

The invention is directed to an isolated genomic polynucleotide, saidpolynucleotide obtainable from human chromosome 11 having a nucleotidesequence at least 95% identical to a sequence selected from the groupconsisting of:

(a) a polynucleotide encoding a polypeptide selected from the groupconsisting of human achaete-scute homolog 2 (HASH2) depicted in SEQ IDNO:1, human SMS3 depicted in SEQ ID NO:2, human tumor suppressingsubtransferable candidate 6 (TSSC6) depicted in SEQ ID NO:3, ribosomalprotein L26 (RIBO26) depicted in SEQ ID NO:4, cluster of differentiationantigen 81 (CD81) depicted in SEQ ID NO:5, and tumor suppressingsubtransferable candidate 4 (TSSC4) depicted in SEQ ID NO:6;

(b) a polynucleotide selected from the group consisting of SEQ ID NO:7which encodes human HASH2 depicted in SEQ ID NO:1, SEQ ID NO:8 whichencodes human SMS3 depicted in SEQ ID NO:2, SEQ ID NO:9 which encodeshuman TSSC6 1 depicted in SEQ ID NO:3, SEQ ID NO:10 which encodesribosomal protein L26 (RIBO26) depicted in SEQ ID NO:4, SEQ ID NO:11which encodes human CD81 depicted in SEQ ID NO:5 and SEQ ID NO:12 whichencodes human TSSC4 depicted in SEQ ID NO:6;

(c) a polynucleotide which is a variant of SEQ ID NOS:7, 8, 9, 10, 11 or12,

(d) a polynucleotide which is an allelic variant of SEQ ID NOS:7, 8, 9,10, 11 or 12:

(e) a polynucleotide which encodes a variant of SEQ ID NOS:1, 2, 3, 4,5, or 6;

(f) a polynucleotide which hybridizes to any one of the polynucleotidesspecified in (a)-(e);

(g) a polynucleotide that is a reverse complement to the polynucleotidesspecified in (a) to (f) and

(h) containing at least 10 transcription factor binding sites selectedfrom the group consisting of AP1FJ_Q2, AP1_C, AP1_Q2, AP1_Q4, AP4_Q5,AP4_Q6, ARNT_(—)01, BRN_(—)01, CDPCR3HD_(—)01, CEBPB_(—)01,CETS1P54_(—)01, CMYB_(—)01, CP2_(—)01, CREB_(—)02, CREB_Q4, CREL_(—)01,DELTAEF1_(—)01, E47_(—)01, FREAC7_(—)01, GATA1_(—)02, GATA1_(—)03,GATA1_(—)04, GATA1_(—)06, GATA2_(—)02, GATA2_(—)03, GATA3_(—)02,GATA3_(—)03, GATA_C, GC_(—)01, GF11_(—)01, HFH2_(—)01, HFH3_(—)01,HFH8_(—)01, IK1_(—)01, IK2_(—)01, LMO2COM_(—)01, LMO2COM_(—)02,LYF1_(—)01, MAX_(—)01, MYCMAX_(—)02, MYOD_(—)01, MYOD_Q6, MZF1_(—)01,NF1_Q6, NFAT_Q6, NKX25_(—)01, NKX25_(—)02, NMYC_(—)01, OCT1_(—)02,PADS_C, RORA1_(—)01, S8_(—)01, SOX5_(—)01, SP1_Q6, STSSC6_(—)01,SRV_(—)02, STAT_(—)01, TATA_(—)01, TCF11_(—)01, USF_(—)01, USF_C, USF_Q6and VMYB_(—)02,

as well as nucleic acid constructs, expression vectors and host cellscontaining these polynucleotide sequences.

The polynucleotides of the present invention may be used for themanufacture of a gene therapy for the prevention, treatment oramelioration of a medical condition by adding an amount of a compositioncomprising said polynucleotide effective to prevent, treat or amelioratesaid medical condition.

The invention is further directed to obtaining these polypeptides by

(a) culturing host cells comprising these sequences under conditionsthat provide for the expression of said polypeptide and

(b) recovering said expressed polypeptide.

The polypeptides obtained may be used to produce antibodies by

(a) optionally conjugating said polypeptide to a carrier protein;

(b) immunizing a host animal with said polypeptide or peptide-carrierprotein conjugate of step (b) with an adjuvant and

(c) obtaining antibody from said immunized host animal.

The invention is further directed to polynucleotides that hybridize tononcoding regions of said polynucleotide sequences as well as antisenseoligonucleotides to these polynucleotides as well as antisense mimetics.The antisense oligonucleotides or mimetics may be used for themanufacture of a medicament for prevention, treatment or amelioration ofa medical condition. The invention is further directed to kitscomprising these polynucleotides and kits comprising these antisenseoligonucleotides or mimetics.

In a specific embodiment, the noncoding regions are transcriptionregulatory regions. The transcription regulatory regions may be used toproduce a heterologous peptide by expressing in a host cell, saidtranscription regulatory region operably linked to a polynucleotideencoding the heterologous polypeptide and recovering the expressedheterologous polypeptide.

The polynucleotides of the present invention may be used to diagnose apathological condition in a subject comprising

(a) determining the presence or absence of a mutation in thepolynucleotides of the present invention and

(b) diagnosing a pathological condition or a susceptibility to apathological condition based on the presence or absence of saidmutation.

The invention is also directed to an isolated polynucleotide from thep15 region of human chromosome 11 selected from the group consisting ofSEQ ID NOS: 13 and 14. SEQ ID NO:13 consists of nucleotide sequenceimmediately preceding the HASH2 gene; SEQ ID NO:14 consists of the gapbetween the RIBO26 and CD81 gene. Both of these polynucleotides arelocated in the imprinted subdomains of 11p15. Oligonucleotides derivedfrom these sequences may be used to identify mutations, duplications,translocations, polysomies and mosaicism associated withBeckwith-Wiedemann syndrome. Furthermore, oligonucleotides derived fromSEQ ID NO:13 may also be used as a marker for the HASH2 gene and SEQ IDNO:14 may be used as a marker for the RIBO26 and/or CD81 gene.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to isolated genomic polynucleotide fragmentsthat encode HASH2, human SMS3, human TSSC6, human RIBO26, human CD81 andhuman TSSC4, which in a specific embodiment are the HASH2, SMS3, TSSC6,RIBO26, CD81 and TSSC4 genes, as well as vectors and hosts containingthese fragments and polynucleotide fragments hybridizing to noncodingregions, as well as antisense oligonucleotides to these fragments.

As defined herein, a “gene” is the segment of DNA involved in producinga polypeptide chain; it includes regions preceding and following thecoding region, as well as intervening sequences (introns) betweenindividual coding segments (exons).

As defined herein “isolated” refers to material removed from itsoriginal environment and is thus altered “by the hand of man” from itsnatural state. An isolated polynucleotide can be part of a vector, acomposition of matter or can be contained within a cell as long as thecell is not the original environment of the polynucleotide.

The polynucleotides of the present invention may be in the form of RNAor in the form of DNA, which DNA includes genomic DNA and synthetic DNA.The DNA may be double-stranded or single-stranded and if single strandedmay be the coding strand or non-coding strand.

The HASH2 gene is 17290 base pairs in length and contains a single exon(see Table 1 below). The HASH2 gene is situated in genomic cloneAC002536 at nucleotides 17081-34370. The SMS3 gene is 25970 base pairsin length and contains 3 exons (Table 2). The SMS3 gene is situated ingenomic clone AC002536 at nucleotides 34371-60340. The TSSC6 gene is30196 base pairs in length and contains 9 exons (Table 3). The TSSC6gene is situated in genomic clone AC002536 at nucleotides 51731-81926.The RIBO26 gene is 21630 base pairs in length and contains a single exon(see Table 4 below for location of the exon). As will be discussed infurther detail below, the RIBO26 gene is situated in genomic cloneAC002536 at nucleotides 77701-99330. The CD81 gene is 21573 base pairsin length and contains 8 exons (Table 5). The CD81 gene begins atnucleotide 120961 in genomic clone AC002536 and extends to nucleotide3640 in the downstream genomic clone AC003693. Clones AC002536 (140977base pairs) and AC003693 (155074 base pairs) have a 2084 base pairoverlap. The TSSC4 gene is 15540 base pairs in length and contains asingle exon (Table 6). The TSSC4 gene is situated in genomic cloneAC003693 at nucleotides 3641-19, 180.

The polynucleotides of the invention have at least a 95% identity andmay have a 96%, 97%, 98% or 99% identity to the polynucleotides depictedin SEQ ID NOS:7, 8, 9, 10, 11 or 12, as well as the polynucleotides inreverse sense orientation, or the polynucleotide sequences encoding theHASH2, SMS3, TSSC6, RIBO26, CD81 or TSSC4 polypeptides depicted in SEQID NOS:1, 2, 3, 4, 5 or 6 respectively.

A polynucleotide having 95% “identity” to a reference nucleotidesequence of the present invention, is identical to the referencesequence except that the polynucleotide sequence may include, onaverage, up to five point mutations per each 100 nucleotides of thereference nucleotide sequence encoding the polypeptide. In other words,to obtain a polynucleotide having a nucleotide sequence at least 95%identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. The query sequence may be an entire sequence, the ORF (openreading frame), or any fragment specified as described herein.

As a practical matter, whether any particular nucleic acid molecule orpolypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to anucleotide sequence of the present invention can be determinedconventionally using known computer programs. A preferred method fordetermining the best overall match between a query sequence (a sequenceof the present invention) and a subject sequence, also referred to as aglobal sequence alignment, can be determined using the FASTDB computerprogram based on the algorithm of Brutlag et al. (Comp. App. Biosci.(1990) 6:237-245). In a sequence alignment the query and subjectsequences are both DNA sequences. An RNA sequence can be compared byconverting U's to T's. The result of said global sequence alignment isin percent identity. Preferred parameters used in a FASTDB alignment ofDNA sequences to calculate percent identity are: Matrix=Unitary,k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization GroupLength=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty=0.05, WindowSize=500 or the length of the subject nucleotide sequence, whichever isshorter.

If the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionmust be made to the results. This is because the FASTDB program does notaccount for 5′ and 3′ truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare 5′ and 3′ of the subject sequence, which are not matched/aligned, asa percent of the total bases of the query sequence. Whether a nucleotideis matched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identify,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This corrected score iswhat is used for the purposes of the present invention. Only basesoutside the 5′ and 3′ bases of the subject sequence, as displayed by theFASTDB alignment, which are not matched/aligned with the query sequenceare calculated for the purposes of manually adjusting the percentidentity score.

For example, a 90 base subject sequence is aligned to a 100 base querysequence to determine percent identity. The deletions occur at the 5′end of the subject sequence and therefore, the FASTDB alignment does notshow a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total numbers of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This time,the deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are made forpurposes of the present invention.

A polypeptide that has an amino acid sequence at least, for example, 95%“identical” to a query amino acid sequence is identical to the querysequence except that the subject polypeptide sequence may include, onaverage, up to five amino acid alterations per each 100 amino acids ofthe query amino acid sequence. In other words, to obtain a polypeptidehaving an amino acid sequence at least 95% identical to a query aminoacid sequence, up to 5% of the amino acid residues in the subjectsequence may be inserted (indels), deleted, or substituted with anotheramino acid. These alterations of the reference sequence may occur at theamino or carboxy terminal positions of the reference amino acid sequenceor anywhere between those terminal positions, interspersed eitherindividually among residues in the referenced sequence or in one or morecontiguous groups within the reference sequence.

A preferred method for determining the best overall match between aquery sequence (a sequence of the present invention) and a subjectsequence, also referred to as a global sequence alignment, can bedetermined using the FASTDB computer program based on the algorithm ofBrutlag et al. (Com. App. Biosci. (1990) 6:237-245). In a sequencealignment, the query and subject sequence are either both nucleotidesequences or both amino acid sequences. The result of said globalsequence alignment is in percent identity. Preferred parameters used ina FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, MismatchPenalty=1, Joining Penalty=20, Randomization Group Length=0, CutoffScore=1, Window Size=sequence length, Gap Penalty=5, Gap SizePenalty=0.05, Window Size=500 or the length of the subject amino acidsequence, whichever is shorter.

If the subject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, a manualcorrection must be made to the results. This is because the FASTDBprogram does not account for N- and C-terminal truncations of thesubject sequence when calculating global percent identity. For subjectsequences truncated at the N- and C-termini, relative to the querysequence, the percent identity is corrected by calculating the number ofresidues of the query sequence that are N- and C-terminal of the subjectsequence, which are not matched/aligned with a corresponding subjectresidue, as a percent of the total bases of the query sequence. Whethera residue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the above FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This finalpercent identity score is what is used for the purposes of the presentinvention. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence.

The invention also encompasses polynucleotides that hybridize to thepolynucleotides depicted in SEQ ID NOS: 7, 8, 9, 10, 11 or 12. Apolynucleotide “hybridizes” to another polynucleotide, when asingle-stranded form of the polynucleotide can anneal to the otherpolynucleotide under the appropriate conditions of temperature andsolution ionic strength (see Sambrook et al., supra). The conditions oftemperature and ionic strength determine the “stringency” of thehybridization. For preliminary screening for homologous nucleic acids,low stringency hybridization conditions, corresponding to a temperatureof 42° C., can be used, e.g., 5×SSC, 0.1% SDS, 0.25% milk, and noformamide; or 40% formamide, 5×SSC, 0.5% SDS). Moderate stringencyhybridization conditions correspond to a higher temperature of 55° C.,e.g., 40% formamide, with 5× or 6×SCC. High stringency hybridizationconditions correspond to the highest temperature of 65° C., e.g., 50%formamide, 5× or 6×SCC. Hybridization requires that the two nucleicacids contain complementary sequences, although depending on thestringency of the hybridization, mismatches between bases are possible.The appropriate stringency for hybridizing nucleic acids depends on thelength of the nucleic acids and the degree of complementation, variableswell known in the art. The greater the degree of similarity or homologybetween two nucleotide sequences, the greater the value of T_(m) forhybrids of nucleic acids having those sequences. The relative stability(corresponding to higher T_(m)) of nucleic acid hybridizations decreasesin the following order: RNA:RNA, DNA:RNA, DNA:DNA.

Polynucleotide and Polypeptide Variants

The invention is directed to both polynucleotide and polypeptidevariants. A “variant” refers to a polynucleotide or polypeptidediffering from the polynucleotide or polypeptide of the presentinvention, but retaining essential properties thereof. Generally,variants are overall closely similar and in many regions, identical tothe polynucleotide or polypeptide of the present invention.

The variants may contain alterations in the coding regions, non-codingregions, or both. Especially preferred are polynucleotide variantscontaining alterations which produce silent substitutions, additions, ordeletions, but do not alter the properties or activities of the encodedpolypeptide. Nucleotide variants produced by silent substitutions due tothe degeneracy of the genetic code are preferred. Moreover, variants inwhich 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or addedin any combination are also preferred.

The invention also encompasses allelic variants of said polynucleotides.An allelic variant denotes any of two or more alternative forms of agene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in polymorphism withinpopulations. Gene mutations can be silent (no change in the encodedpolypeptide) or may encode polypeptides having altered amino acidsequences. An allelic variant of a polypeptide is a polypeptide encodedby an allelic variant of a gene.

The amino acid sequences of the variant polypeptides may differ from theamino acid sequences depicted in SEQ ID NOS:1, 2, 3, 4, 5 or 6 by aninsertion or deletion of one or more amino acid residues and/or thesubstitution of one or more amino acid residues by different amino acidresidues. Preferably, amino acid changes are of a minor nature, that isconservative amino acid substitutions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof one to about 30 amino acids; small amino- or carboxyl-terminalextensions, such as an amino-terminal methionine residue; a small linkerpeptide of up to about 20-25 residues; or a small extension thatfacilitates purification by changing net charge or another function,such as a poly-histidine tract, an antigenic epitope or a bindingdomain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions which do not generally alter the specific activityare known in the art and are described, for example, by H. Neurath andR. L. Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, as well as these in reverse.

Noncoding Regions

The invention is further directed to polynucleotide fragments containingor hybridizing to noncoding regions of the HASH2, SMS3, TSSC6, RIBO26,CD81 and TSSC4 genes. These include but are not limited to an intron, a5′-non-coding region, a 3′-non-coding region and splice junctions (seeTables 1-6), as well as transcription factor binding sites (see Table7). The polynucleotide fragments may be a short polynucleotide fragmentwhich is between about 8 nucleotides to about 40 nucleotides in length.Such shorter fragments may be useful for diagnostic purposes. Such shortpolynucleotide fragments are also preferred with respect topolynucleotides containing or hybridizing to polynucleotides containingsplice junctions. Alternatively larger fragments, e.g., of about 50,150, 500, 600 or about 2000 nucleotides in length may be used.

TABLE 1 Exon/Intron Regions of the human achaete-scute homolog 2 (HASH2)gene, 17290 bp, reference cDNA accession number U77629; reverse strandcoding. Exon Location (nucleotide no./amino acid no.) 1 7031-7609 193-1 stop codon 7028-7030

TABLE 2 Exon/Intron Regions of the human SMS3 gene, 25970 bp, referencecDNA accession number AB029488; reverse strand coding. Exon Location(nucleotide no./amino acid no.) 3 18962-19210 132-50  2 20023-2011849-18 1 21261-21311  1-17 stop codon 18959-18961

TABLE 3 Exon/Intron Regions of the human tumor suppressingsubtransferable candidate 6 (TSSC6) gene, 30196 bp, reference cDNAaccession number NM_005705; plus strand coding. Exon Location(nucleotide no./amino acid no.) 1 5011-5100  1-30 2 6249-6347 31-63 310879-10953 64-88 4 15797-15898  89-122 5 16628-16714 123-151 618372-18455 152-179 7 18719-18811 180-210 8 19488-19664 211-270 920005-20064 271-290 stop codon 20065-20067

TABLE 4 Exon/Intron Regions of the human ribosomal protein L26 gene,21630 bp, reference cDNA accession number AF083248; reverse strandcoding. Exon Location (nucleotide no./amino acid no.) 1 11490-11924145-1  stop codon 11487-11489

TABLE 5 Exon/Intron Region of the human CD81 gene, 37113 bp, referenceaccession number NM_004356; plus strand coding. Exon Location(nucleotide no./amino acid no.) 1 10471-10536  1-22 2 23333-23446 23-603 27015-27113 61-93 4 27893-27964  94-117 5 28334-28441 118-153 628790-28891 154-187 7 29549-29635 188-216 8 29725-29784 217-236 stopcodon 29785-29787

TABLE 6 Exon/Intro Region of the human tumor suppressing subtransferablecandidate 4 (TSSC4) gene, 15540 bp, reference cDNA accession numberNM_005706; plus strand coding. Exon Location (nucleotide no./amino acidno.) 1 13982-14968  1-329 stop codon 14969-14971

TABLE 7 TRANSCRIPTION FACTOR BINDING SITES BINDING SITES HASH2 SMS3TSSC6 RIBO26 CD81 TSSC4 AP1FJ_Q2 14 8 10 16 AP1_C 4 6 8 10 8 AP1_Q2 4 75 10 6 AP1_Q4 4 5 5 AP4_Q5 30 44 55 12 71 AP4_Q6 14 22 26 4 34 ARNT_01 74 6 BRN2_01 5 4 CDPCR3HD_01 5 8 CEBPB_01 9 5 13 4 CETS1P54_01 5 CMYB_014 CP2_01 4 5 CREB_02 4 CREB_Q4 4 CREL_01 5 11 11 7 DELTAEF1_01 42 49 6757 84 E47_01 6 17 FREAC7_01 4 6 GATA1_02 6 7 6 9 11 GATA1_03 8 7 4 15 5GATA1_04 9 16 10 11 10 GATA1_05 5 7 5 GATA1_06 4 7 GATA2_02 7 12 6 8 4GATA2_03 6 GATA3_02 4 6 GATA3_03 4 GATA_C 6 13 5 7 7 GC_01 7 GFI1_01 6HFH2_01 4 4 HFH3_01 5 9 7 4 HFH8_01 4 5 IK1_01 4 IK2_01 22 24 34 33 56LMO2COM_01 21 33 41 18 57 7 LMO2COM_02 13 15 10 11 14 LYF1_01 5 7 4 6MAX_01 4 MYCMAX_02 4 MYOD_01 4 MYOD_Q6 13 13 22 5 34 11 MZF1_01 73 106136 63 211 21 NF1_Q6 5 6 6 NFAT_Q6 23 33 20 39 16 NKX25_01 6 4 4 7 4NKX25_02 4 NMYC_01 14 15 4 10 OCT1_02 6 PADS_C 6 4 RORA1_01 4 S8_01 5 2515 23 7 SOX5_01 5 9 5 8 11 SP1_Q6 6 11 SRY_02 4 6 9 STAT_01 5 5 TATA_016 TCF11_01 24 27 27 43 43 9 USF_01 14 16 4 10 12 4 USF_C 14 16 4 10 12 6USF_Q6 10 6 VMYB_02 9 5 4 11 Abbreviations: HASH2, human achaete-scutehomolog 2; TSSC6, tumor suppressing subtransferable candidate 6; RIBO26,ribosomal protein L26; CD81, cluster of differentiation antigen 81; andTSSC4, tumor suppressing subtransferable candidate 4.

In a specific embodiment, such noncoding sequences are expressioncontrol sequences. These include but are not limited to DNA regulatorysequences, such as promoters, enhancers, repressors, terminators, andthe like, that provide for the regulation of expression of a codingsequence in a host cell. In eukaryotic cells, polyadenylation signalsare also control sequences.

In a more specific embodiment of the invention, the expression controlsequences may be operatively linked to a polynucleotide encoding aheterologous polypeptide. Such expression control sequences may be about50-200 nucleotides in length and specifically about 50, 100, 200, 500,600, 1000 or 2000 nucleotides in length. A transcriptional controlsequence is “operatively linked” to a polynucleotide encoding aheterologous polypeptide sequence when the expression control sequencecontrols and regulates the transcription and translation of thatpolynucleotide sequence. The term “operatively linked” includes havingan appropriate start signal (e.g., ATG) in front of the polynucleotidesequence to be expressed and maintaining the correct reading frame topermit expression of the DNA sequence under the control of theexpression control sequence and production of the desired productencoded by the polynucleotide sequence. If a gene that one desires toinsert into a recombinant DNA molecule does not contain an appropriatestart signal, such a start signal can be inserted upstream (5′) of andin reading frame with the gene.

The invention is further directed to antisense oligonucleotides andmimetics to these polynucleotide sequences. Antisense technology can beused to control gene expression through triple-helix formation orantisense DNA or RNA, both of which methods are based on binding of apolynucleotide to DNA or RNA. For example, the 5′ coding portion of thepolynucleotide sequence, which encodes the mature polypeptides of thepresent invention, is used to design an antisense RNA oligonucleotide offrom about 10 to 40 base pairs in length. A DNA oligonucleotide isdesigned to be complementary to a region of the gene involved intranscription or RNA processing (triple helix (see Lee et al., Nucl.Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); andDervan et al., Science, 251: 1360 (1991)), thereby preventingtranscription and the production of said polypeptides.

Expression of Polypeptides Isolated Polynucleotide Sequences

The human chromosome 11 genomic clone of accession number AC002536 hasbeen discovered to contain the HASH2 gene, the SMS3 gene, the TSSC6gene, the RIBO26, part of the CD81 gene by Genscan analysis (Burge etal., 1997, J. Mol. Biol. 268:78-94), BLAST2 and TBLASTN analysis(Altschul et al., 1997, Nucl. Acids Res. 25:3389-3402), in which thesequence of AC002536 was compared to the HASH2 cDNA sequence, accessionnumber U77629, the human SMS3 cDNA sequence accession number AB029488,TSSC6 cDNA sequence accession number NM_(—)005705, and the RIBO26 cDNAsequence, accession number AF083248. The remainder of the CD81 gene andthe TSSC4 gene were found by similar means in the downstream cloneAC003693. The accession numbers for the CD81 and TSSC4 cDNAs are,respectively, NM_(—)004356 and NM_(—)005706.

The cloning of the nucleic acid sequences of the present invention fromsuch genomic DNA can be effected, e.g., by using the well knownpolymerase chain reaction (PCR) or antibody screening of expressionlibraries to detect cloned DNA fragments with shared structuralfeatures. See, e.g., Innis et al., 1990, PCR: A Guide to Methods andApplication, Academic Press, New York. Other nucleic acid amplificationprocedures such as ligase chain reaction (LCR), ligated activatedtranscription (LAT) and nucleic acid sequence-based amplification(NASBA) or long range PCR may be used. In a specific embodiment, 5′- or3′-non-coding portions of each gene may be identified by methodsincluding but are not limited to, filter probing, clone enrichment usingspecific probes and protocols similar or identical to 5′- and 3′-“RACE”protocols which are well known in the art. For instance, a methodsimilar to 5′-RACE is available for generating the missing 5′-end of adesired full-length transcript. (Fromont-Racine et al., 1993, Nucl.Acids Res. 21:1683-1684).

Once the DNA fragments are generated, identification of the specific DNAfragment containing the desired HASH2 gene, the SMS3 gene, the TSSC6gene, the RIBO26 gene, the CD81 gene, the TSSC4 gene, SEQ ID NO:13 orSEQ ID NO:14 may be accomplished in a number of ways. For example, if anamount of a portion of the HASH2 gene, the SMS3 gene, the TSSC6 gene,the RIBO26 gene, the CD81 gene or the TSSC4 gene or its specific RNA, ora fragment thereof, is available and can be purified and labeled, thegenerated DNA fragments may be screened by nucleic acid hybridization tothe labeled probe (Benton and Davis, 1977, Science 196:180; Grunsteinand Hogness, 1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961). The presentinvention provides such nucleic acid probes, which can be convenientlyprepared from the specific sequences disclosed herein, e.g., ahybridizable probe having a nucleotide sequence corresponding to atleast a 10, and preferably a 15, nucleotide fragment of the sequencesdepicted in SEQ ID NOS:7, 8, 9, 10, 11, 12, 13 or 14. Preferably, afragment is selected that is highly unique to the polypeptides of theinvention. Those DNA fragments with substantial homology to the probewill hybridize. As noted above, the greater the degree of homology, themore stringent hybridization conditions can be used. In one embodiment,low stringency hybridization conditions are used to identify ahomologous HASH2, SMS3, TSSC6, or RIBO26 polynucleotide. However, in apreferred aspect, and as demonstrated experimentally herein, a nucleicacid encoding a polypeptide of the invention will hybridize to a nucleicacid derived from the polynucleotide sequence depicted in SEQ ID NOS:7,8, 9, 10, 11 or 12 or a hybridizable fragment thereof, under moderatelystringent conditions; more preferably, it will hybridize under highstringency conditions.

Alternatively, the presence of the gene may be detected by assays basedon the physical, chemical, or immunological properties of its expressedproduct. For example, cDNA clones, or DNA clones which hybrid-select theproper mRNAs, can be selected which produce a protein that, e.g., hassimilar or identical electrophoretic migration, isoelectric focusingbehavior, proteolytic digestion maps, or antigenic properties as knownfor the HASH2, SMS3, the TSSC6, RIBO26, CD81 or TSSC4 polypeptide.

A gene encoding HASH2, SMS3, TSSC6, RIBO26, CD81 or TSSC4 polypeptidecan also be identified by mRNA selection, i.e., by nucleic acidhybridization followed by in vitro translation. In this procedure,fragments are used to isolate complementary mRNAs by hybridization.Immunoprecipitation analysis or functional assays of the in vitrotranslation products of the products of the isolated mRNAs identifiesthe mRNA and, therefore, the complementary DNA fragments, that containthe desired sequences.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide sequence containing the exon/intron segments of theHASH2 gene (nucleotides 7028-7609 of SEQ ID NO:7), SMS3 gene(nucleotides 18959-21311 of SEQ ID NO:8), TSSC6 gene (nucleotides5011-20067 of SEQ ID NO:9), RIBO26 gene (nucleotides 11487-11924 of SEQID NO:10), CD81 gene (nucleotides 10471-29787 of SEQ ID NO:11) or TSSC4gene (nucleotides 13982-14971 of SEQ ID NO:12) operably linked to one ormore control sequences which direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences. Expression will be understood to include any stepinvolved in the production of the polypeptide including, but not limitedto, transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

The invention is further directed to a nucleic acid construct comprisingexpression control sequences derived from SEQ ID NOS: 7, 8, 9, 10, 11 or12 and a heterologous polynucleotide sequence.

“Nucleic acid construct” is defined herein as a nucleic acid molecule,either single- or double-stranded, which is isolated from a naturallyoccurring gene or which has been modified to contain segments of nucleicacid which are combined and juxtaposed in a manner which would nototherwise exist in nature. The term nucleic acid construct is synonymouswith the term expression cassette when the nucleic acid constructcontains all the control sequences required for expression of a codingsequence of the present invention. The term “coding sequence” is definedherein as a portion of a nucleic acid sequence which directly specifiesthe amino acid sequence of its protein product. The boundaries of thecoding sequence are generally determined by a ribosome binding site(prokaryotes) or by the ATG start codon (eukaryotes) located justupstream of the open reading frame at the 5′ end of the mRNA and atranscription terminator sequence located just downstream of the openreading frame at the 3′ end of the mRNA. A coding sequence can include,but is not limited to, DNA, cDNA, and recombinant nucleic acidsequences.

The isolated polynucleotide of the present invention may be manipulatedin a variety of ways to provide for expression of the polypeptide.Manipulation of the nucleic acid sequence prior to its insertion into avector may be desirable or necessary depending on the expression vector.The techniques for modifying nucleic acid sequences utilizingrecombinant DNA methods are well known in the art.

The control sequence may be an appropriate promoter sequence, a nucleicacid sequence which is recognized by a host cell for expression of thenucleic acid sequence. The promoter sequence contains transcriptionalcontrol sequences which regulate the expression of the polynucleotide.The promoter may be any nucleic acid sequence which showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, the prokaryotic beta-lactamase gene (VIIIa-Komaroff et al.,1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as the tacpromoter (DeBoer et al., 1983, Proc. Natl. Acad. of Sciences USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Scientific American, 1980, 242: 74-94; and inSambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes encoding Aspergillusoryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillusniger neutral alpha-amylase, Aspergillus niger acid stablealpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase(glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulansacetamidase, Fusarium oxysporum trypsin-like protease (WO 96/00787),NA2-tpi (a hybrid of the promoters from the genes encoding Aspergillusniger neutral alpha-amylase and Aspergillus oryzae triose phosphateisomerase), and mutant, truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the Saccharomycescerevisiae enolase (ENO-1) gene, the Saccharomyces cerevisiaegalactokinase gene (GAL1), the Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP),and the Saccharomyces cerevisiae 3-phosphoglycerate kinase gene. Otheruseful promoters for yeast host cells are described by Romanos et al.,1992, Yeast 8: 423-488.

Eukaryotic promoters may be obtained from the genomes of viruses such aspolyoma virus, fowlpox virus, adenovirus, bovine papilloma virus, aviansarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus andSV40. Alternatively, heterologous mammalian promoters, such as the actinpromoter or immunoglobulin promoter may be used.

The constructs of the invention may also include enhancers. Enhancersare cis-acting elements of DNA, usually from about 10 to about 300 bpthat act on a promoter to increase its transcription. Enhancers fromglobin, elastase, albumin, alpha-fetoprotein, and insulin enhancers maybe used. However, an enhancer from a virus may be used; examples includeSV40 on the late side of the replication origin, the cytomegalovirusearly promoter enhancer, the polyoma enhancer on the late side of thereplication origin and adenovirus enhancers.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleic acid sequence encoding the polypeptide. Any leadersequence that is functional in the host cell of choice may be used inthe present invention.

The control sequence may also be a polyadenylation sequence, a sequencewhich is operably linked to the 3′ terminus of the nucleic acid sequenceand which, when transcribed, is recognized by the host cell as a signalto add polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention.

The control sequence may also be a signal peptide coding region, whichcodes for an amino acid sequence linked to the amino terminus of thepolypeptide which can direct the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not normallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to obtain enhanced secretion of thepolypeptide. However, any signal peptide coding region which directs theexpressed polypeptide into the secretory pathway of a host cell ofchoice may be used in the present invention.

The control sequence may also be a propeptide coding region, which codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpro-polypeptide (or a zymogen in some cases). A pro-polypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepro-polypeptide. The propeptide coding region may be obtained from theBacillus subtilis alkaline protease gene (aprE), the Bacillus subtilisneutral protease gene (nprT), the Saccharomyces cerevisiae alpha-factorgene, the Rhizomucor miehei aspartic proteinase gene, or theMyceliophthora thermophila laccase gene (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems would include thelac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1system may be used. In filamentous fungi, the TAKA alpha-amylasepromoter, Aspergillus niger glucoamylase promoter, and the Aspergillusoryzae glucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene which is amplified in the presence of methotrexate, andthe metallothionein genes which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the polypeptide would beoperably linked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a nucleic acid sequence of the present invention, a promoter,and transcriptional and translational stop signals. The various nucleicacid and control sequences described above may be joined together toproduce a recombinant expression vector which may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe nucleic acid sequence encoding the polypeptide at such sites.Alternatively, the polynucleotide of the present invention may beexpressed by inserting the nucleic acid sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about the expression of the nucleic acid sequence. Thechoice of the vector will typically depend on the compatibility of thevector with the host cell into which the vector is to be introduced. Thevectors may be linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophs,and the like. Examples of bacterial selectable markers are the dal genesfrom Bacillus subtilis or Bacillus licheniformis, or markers whichconfer antibiotic resistance such as ampicillin, kanamycin,chloramphenicol or tetracycline resistance. Suitable markers for yeasthost cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. An exampleof suitable selectable markers for mammalian cells are those that enablethe identification of cells competent to take of the nucleic acids ofthe present invention, such as DHFRor thymidine kinase. An appropriatehost cell when wild-type DHFR is employed is the CHO cell line deficientin DHFR activity, prepared and propagated as described by Urlaub et al.,Proc. Natl. Acad. Sci. USA, 77:4216 (1980).

The vectors of the present invention preferably contain an element(s)that permits stable integration of the vector into the host cell genomeor autonomous replication of the vector in the cell independent of thegenome of the cell.

For integration into the host cell genome, the vector may rely on thepolynucleotide sequence encoding the polypeptide or any other element ofthe vector for stable integration of the vector into the genome byhomologous or nonhomologous recombination. Alternatively, the vector maycontain additional nucleic acid sequences for directing integration byhomologous recombination into the genome of the host cell. Theadditional polynucleotide sequences enable the vector to be integratedinto the host cell genome at a precise location(s) in the chromosome(s).To increase the likelihood of integration at a precise location, theintegrational elements should preferably contain a sufficient number ofnucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500base pairs, and most preferably 800 to 1,500 base pairs, which arehighly homologous with the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleic acid sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are theorigins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAMB1permitting replication in Bacillus. Examples of origins of replicationfor use in a yeast host cell are the 2 micron origin of replication,ARS1, ARS4, the combination of ARS1 and CEN3, and the combination ofARS4 and CEN6. The origin of replication may be one having a mutationwhich makes its functioning temperature-sensitive in the host cell (see,e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA75: 1433).

More than one copy of a polynucleotide sequence of the present inventionmay be inserted into the host cell to increase production of the geneproduct. An increase in the copy number of the polynucleotide sequencecan be obtained by integrating at least one additional copy of thesequence into the host cell genome or by including an amplifiableselectable marker gene with the nucleic acid sequence where cellscontaining amplified copies of the selectable marker gene, and therebyadditional copies of the nucleic acid sequence, can be selected for bycultivating the cells in the presence of the appropriate selectableagent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga nucleic acid sequence of the invention, which are advantageously usedin the recombinant production of the polypeptides. A vector comprising anucleic acid sequence of the present invention is introduced into a hostcell so that the vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector as described earlier. The term“host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The host cell may be a unicellular microorganism, e.g., a prokaryote, ora non-unicellular microorganism, e.g., a eukaryote. Useful unicellularcells are bacterial cells such as gram positive bacteria including, butnot limited to, a Bacillus cell, or a Streptomyces cell, e.g.,Streptomyces lividans or Streptomyces murinus, or gram negative bacteriasuch as E. coli and Pseudomonas sp.

The introduction of a vector into a bacterial host cell may, forinstance, be effected by protoplast transformation (see, e.g., Chang andCohen, 1979, Molecular General Genetics 168: 111-115), using competentcells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5771-5278).

The host cell may be a eukaryote, such as a mammalian cell (e.g., humancell), an insect cell, a plant cell or a fungal cell. Mammalian hostcells that could be used include but are not limited to human Hela,embryonic kidney cells (293), lung cells, H9 and Jurkat cells, mouseNIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse Lcells and Chinese Hamster ovary (CHO) cells. These cells may betransfected with a vector containing a transcriptional regulatorysequence, a protein coding sequence and transcriptional terminationsequences by lipid-mediated, calcium phosphate mediated or DEAE-dextranmediated transfection (reviewed in Sambrook and Russell, MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 2001). Alternatively, the polypeptide can beexpressed in stable cell lines containing the polynucleotide integratedinto a chromosome. The co-transfection with a selectable marker such asdhfr, gpt, neomycin, hygromycin allows the identification and isolationof the transfected cells. The polynucleotide may be directly introducedinto the eukaryotic cell via electroporation, bolistics, or polybrene(reviewed in Sambrook and Russell, supra).

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (asdefined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary ofThe Fungi, 8th edition, 1995, CAB International, University Press,Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al.,1995, supra, page 171) and all mitosporic fungi (Hawksworth et al.,1995, supra). The fungal host cell may also be a yeast cell. “Yeast” asused herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980). The fungal host cell may also be a filamentous fungalcell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are characterized by a mycelial wallcomposed of chitin, cellulose, glucan, chitosan, mannan, and othercomplex polysaccharides. Vegetative growth is by hyphal elongation andcarbon catabolism is obligately aerobic. In contrast, vegetative growthby yeasts such as Saccharomyces cerevisiae is by budding of aunicellular thallus and carbon catabolism may be fermentative.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus host cells are described in EP 238 023 andYelton et al., 1984, Proceedings of the National Academy of Sciences USA81: 1470-1474. Suitable methods for transforming Fusarium species aredescribed by Malardier et al., 1989, Gene 78: 147-156 and WO 96/00787.Yeast may be transformed using the procedures described by Becker andGuarente, In Abelson, J. N. and Simon, M. I., editors, Guide to YeastGenetics and Molecular Biology, Methods in Enzymology, Volume 194, pp182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal ofBacteriology 153: 163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci.USA 75: 1920.

Methods of Production

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide; and(b) recovering the polypeptide.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods known in the art. For example, the cell may becultivated by shake flask cultivation, small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermentors performed in asuitable medium and under conditions allowing the polypeptide to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, the presence of the HASH2 andRIBO26 protein may be detected using standard transcription assays. Thepresence of TSSC4 and TSSC6 may be detected by assaying for tumorsuppressor activity in rhabdomyosarcoma cells (Koi et al., 1993, Science260:361-364). The presence of CD81 may be detected by assaying forbinding to E2 hepatitis C protein (Allander et al., 2000, J. Gen. Virol.81:2451-2459).

The resulting polypeptide may be recovered by methods known in the art.For example, the polypeptide may be recovered from the nutrient mediumby conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989).

Antibodies

According to the invention, the HASH2, SMS3, TSSC6, RIBO26, CD81 orTSSC4 polypeptides produced according to the method of the presentinvention may be used as an immunogen to generate any of theseantibodies. Such antibodies include but are not limited to polyclonal,monoclonal, chimeric, single chain, Fab fragments, and an Fab expressionlibrary.

Various procedures known in the art may be used for the production ofantibodies. For the production of antibody, various host animals can beimmunized by injection with the polypeptide thereof, including but notlimited to rabbits, mice, rats, sheep, goats, etc. In one embodiment,the polypeptide or fragment thereof can optionally be conjugated to animmunogenic carrier, e.g., bovine serum albumin (BSA) or keyhole limpethemocyanin (KLH). Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

For preparation of monoclonal antibodies directed toward the HASH2,SMS3, TSSC6, RIBO26, CD81 or TSSC4 polypeptide, any technique thatprovides for the production of antibody molecules by continuous celllines in culture may be used. These include but are not limited to thehybridoma technique originally developed by Kohler and Milstein (1975,Nature 256:495-497), as well as the trioma technique, the human B-cellhybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96). In an additional embodiment of the invention,monoclonal antibodies can be produced in germ-free animals utilizingrecent technology (PCT/US90/02545). According to the invention, humanantibodies may be used and can be obtained by using human hybridomas(Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030) or bytransforming human B cells with EBV virus in vitro (Cole et al., 1985,in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96).In fact, according to the invention, techniques developed for theproduction of “chimeric antibodies” (Morrison et al., 1984, J.Bacteriol. 159-870; Neuberger et al., 1984, Nature 312:604-608; Takedaet al., 1985, Nature 314:452-454) by splicing the genes from a mouseantibody molecule specific for the HASH2, SMS3, TSSC6, RIBO26, CD81 orTSSC4 polypeptide together with genes from a human antibody molecule ofappropriate biological activity can be used; such antibodies are withinthe scope of this invention.

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778) can be adapted toproduce polypeptide-specific single chain antibodies. An additionalembodiment of the invention utilizes the techniques described for theconstruction of Fab expression libraries (Huse et al., 1989, Science246:1275-1281) to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity for the HASH₂, SMS3, TSSC6,RIBO26, CD81 or TSSC4 polypeptides.

Antibody fragments which contain the idiotype of the antibody moleculecan be generated by known techniques. For example, such fragmentsinclude but are not limited to: the F(ab′)2 fragment which can beproduced by pepsin digestion of the antibody molecule; the Fab′fragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂, fragment, and the Fab fragments which can be generated bytreating the antibody molecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g., radioimmunoassay,ELISA (enzyme-linked immunosorbent assay), “sandwich” immunoassays,immunoradiometric assays, gel diffusion precipitin reactions,immunodiffusion assays, in situ immunoassays (using colloidal gold,enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention. For example, to select antibodies which recognize aspecific epitope of a particular polypeptide, one may assay generatedhybridomas for a product which binds to a particular polypeptidefragment containing such epitope. For selection of an antibody specificto a particular polypeptide from a particular species of animal, one canselect on the basis of positive binding with the polypeptide expressedby or isolated from cells of that species of animal.

Immortal, antibody-producing cell lines can also be created bytechniques other than fusion, such as direct transformation of Blymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus.See, e.g., M. Schreier et al., “Hybridoma Techniques” (1980); Hammerlinget al., “Monoclonal Antibodies And T-cell Hybridomas” (1981); Kennett etal., “Monoclonal Antibodies” (1980); see also U.S. Pat. Nos. 4,341,761;4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917; 4,472,500;4,491,632; 4,493,890.

Uses of Polynucleotides Diagnostics

Polynucleotides containing noncoding regions of SEQ ID NOS:7, 8, 9, 10,11, 12, 13 or 14 may be used as probes for detecting mutations fromsamples from a patient. Genomic DNA may be isolated from the patient. Amutation(s) may be detected by Southern blot analysis, specifically byhybridizing restriction digested genomic DNA to various probes andsubjecting to agarose electrophoresis. Alternatively, thesepolynucleotides may be used as PCR primers and be used to amplify thegenomic DNA isolated from the patients. Additionally, primers may beobtained by routine or long range PCR that yield products containingcontiguous intron/exon sequence and products containing more than oneexon with intervening intron. The sequence of the amplified genomic DNAfrom the patient may be determined using methods known in the art. Suchprobes may be between 10-100 nucleotides in length and may preferably bebetween 20-50 nucleotides in length. Specifically, probes derived fromSEQ ID NOS: 13 or 14 may be used to identify mutations duplications,translocations, polysomies and mosaicism associated withBeckwith-Wiedemann syndrome.

Thus the invention is thus directed to kits comprising thesepolynucleotide probes. In a specific embodiment, these probes arelabeled with a detectable substance.

Antisense Oligonucleotides and Mimetics

The antisense oligonucleotides or mimetics of the present invention maybe used to decrease levels of a polypeptide. For example, HASH2 isrequired for development of the trophoblast. Therefore, the HASH2antisense oligonucleotides of the present invention could be used as anantifertility agent. RIBO26 is expressed in abundance in small celltumors of the lung. RIBO26 antisense sequences could be used to inhibitsmall cell tumor growth. CD81 plays a role in T cell activation, and itsantisense sequences may help control autoimmune disorders in which Tcell activation is uncontrolled. CD81 also binds the human hepatitis Cvirus; thus CD81 antisense sequences may, by reducing CD81 expression,reduce the infectivity of the human hepatitis C virus. The TSSC4 and 6proteins act as tumor suppressors. Therefore, antisense sequences mayact as antiapoptosis agents.

The HASH2, SMS3, TSSC6, RIBO26, CD81 and TSSC4 genes are all situated ina region of chromosome 11 known to be associated with theBeckwith-Wiedemann Syndrome. Thus, antisense sequences of any of thesesix genes may provide means of managing patients with theBeckwith-Wiedemann Syndrome. Furthermore, antisense oligonucleotides ofSEQ ID NOS:13 or 14 may be used for the same purpose.

The antisense oligonucleotides of the present invention may beformulated into pharmaceutical compositions. These compositions may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.

Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable.

Compositions and formulations for oral administration include powders orgranules, suspensions or solutions in water or non-aqueous media,capsules, sachets or tablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, liquid syrups, soft gels, suppositories, and enemas. Thecompositions of the present invention may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsmay further contain substances which increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention, the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀ as found to be effective in vitroand in vivo animal models.

In general, dosage is from 0.01 ug to 10 g per kg of body weight, andmay be given once or more daily, weekly, monthly or yearly, or even onceevery 2 to 20 years. Persons of ordinary skill in the art can easilyestimate repetition rates for dosing based on measured residence timesand concentrations of the drug in bodily fluids or tissues. Followingsuccessful treatment, it may be desirable to have the patient undergomaintenance therapy to prevent the recurrence of the disease state,wherein the oligonucleotide is administered in maintenance doses,ranging from 0.01 ug to 10 g per kg of body weight, once or more daily,to once every 20 years.

Gene Therapy

As noted above, HASH2 is necessary for development of the trophoblast,RIBO26 is a component of the ribosome, TSSC6 and TSSC4 are involved inrepressing tumor growth, and CD81 is involved in T cell activation.Therefore, the HASH2 gene may be used to treat some forms ofinfertility. The CD81 gene may be used in patients whose ability toactivate T cells is impaired. CD81 also binds the human hepatitis Cvirus, thus gene therapy designed to yield a secretable form of CD81may, by binding the virus in an excretable form, reduce the spread ofhepatitis C. Given the tumor suppressing actions of TSSC6 and TSSC4,their genes may be used to prevent tumor growth. RIBO26 may be used totreat disorders in which ribosome assembly is defective. The SMS3 geneis situated within the Beckwith-Wiedemann Syndrome locus and may thus beuseful for treatment of patients in which the SMS3 gene isnonfunctional.

As described herein, the polynucleotide of the present invention may beintroduced into a patient's cells for therapeutic uses. As will bediscussed in further detail below, cells can be transfected using anyappropriate means, including viral vectors, as shown by the example,chemical transfectants, or physico-mechanical methods such aselectroporation and direct diffusion of DNA. See, for example, Wolff,Jon A, et al., “Direct gene transfer into mouse muscle in vivo,”Science, 247, 1465-1468, 1990; and Wolff, Jon A, “Human dystrophinexpression in mdx mice after intramuscular injection of DNA constructs,”Nature, 352, 815-818, 1991. As used herein, vectors are agents thattransport the gene into the cell without degradation and include apromoter yielding expression of the gene in the cells into which it isdelivered. As will be discussed in further detail below, promoters canbe general promoters, yielding expression in a variety of mammaliancells, or cell specific, or even nuclear versus cytoplasmic specific.These are known to those skilled in the art and can be constructed usingstandard molecular biology protocols. Vectors have been divided into twoclasses:

a) Biological agents derived from viral, bacterial or other sources.

b) Chemical physical methods that increase the potential for geneuptake, directly introduce the gene into the nucleus or target the geneto a cell receptor.

Biological Vectors

Viral vectors have higher transaction (ability to introduce genes)abilities than do most chemical or physical methods to introduce genesinto cells. Vectors that may be used in the present invention includeviruses, such as adenoviruses, adeno associated virus (AAV), vaccinia,herpesviruses, baculoviruses and retroviruses, bacteriophages, cosmids,plasmids, fungal vectors and other recombination vehicles typically usedin the art which have been described for expression in a variety ofeukaryotic and prokaryotic hosts, and may be used for gene therapy aswell as for simple protein expression. Polynucleotides are inserted intovector genomes using methods well known in the art.

Retroviral vectors are the vectors most commonly used in clinicaltrials, since they carry a larger genetic payload than other viralvectors. However, they are not useful in non-proliferating cells.Adenovirus vectors are relatively stable and easy to work with, havehigh titers, and can be delivered in aerosol formulation. Pox viralvectors are large and have several sites for inserting genes, they arethermostable and can be stored at room temperature.

Examples of promoters are SP6, T4, T7, SV40 early promoter,cytomegalovirus (CMV) promoter, mouse mammary tumor virus (MMTV)steroid-inducible promoter, Moloney murine leukemia virus (MMLV)promoter, phosphoglycerate kinase (PGK) promoter, and the like.Alternatively, the promoter may be an endogenous adenovirus promoter,for example the E1 a promoter or the Ad2 major late promoter (MLP).Similarly, those of ordinary skill in the art can construct adenoviralvectors utilizing endogenous or heterologous poly A addition signals.Plasmids are not integrated into the genome and the vast majority ofthem are present only from a few weeks to several months, so they aretypically very safe. However, they have lower expression levels thanretroviruses and since cells have the ability to identify and eventuallyshut down foreign gene expression, the continuous release of DNA fromthe polymer to the target cells substantially increases the duration offunctional expression while maintaining the benefit of the safetyassociated with non-viral transfections.

Chemical/Physical Vectors

Other methods to directly introduce genes into cells or exploitreceptors on the surface of cells include the use of liposomes andlipids, ligands for specific cell surface receptors, cell receptors, andcalcium phosphate and other chemical mediators, microinjections directlyto single cells, electroporation and homologous recombination. Liposomesare commercially available from Gibco BRL, for example, asLIPOFECTIN{umlaut over ( )} and LIPOFECTACE{umlaut over ( )}, which areformed of cationic lipids such as N-[1-(2,3dioleyloxy)-propyl]-n,n,n-trimethylammonium chloride (DOTMA) anddimethyl dioctadecylammonium bromide (DDAB). Numerous methods are alsopublished for making liposomes, known to those skilled in the art.

For example, Nucleic acid-Lipid Complexes—Lipid carriers can beassociated with naked nucleic acids (e.g., plasmid DNA) to facilitatepassage through cellular membranes. Cationic, anionic, or neutral lipidscan be used for this purpose. However, cationic lipids are preferredbecause they have been shown to associate better with DNA which,generally, has a negative charge. Cationic lipids have also been shownto mediate intracellular delivery of plasmid DNA (Felgner and Ringold,Nature 337:387 (1989)). Intravenous injection of cationic lipid-plasmidcomplexes into mice has been shown to result in expression of the DNA inlung (Brigham et al., Am. J. Med. Sci. 298:278 (1989)). See also, Osakaet al., J. Pharm. Sci. 85(6):612-618 (1996); San et al., Human GeneTherapy 4:781-788 (1993); Senior et al., Biochemica et Biophysica Acta1070:173-179 (1991); Kabanov and Kabanov, Bioconjugate Chem. 6:7-20(1995); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Behr, J-P.,Bioconjugate Chem 5:382-389 (1994); Behr et al., Proc. Natl. Acad. Sci.,USA 86:6982-6986 (1989); and Wyman et al., Biochem. 36:3008-3017 (1997).

Cationic lipids are known to those of ordinary skill in the art.Representative cationic lipids include those disclosed, for example, inU.S. Pat. No. 5,283,185; and e.g., U.S. Pat. No. 5,767,099. In apreferred embodiment, the cationic lipid is N⁴-spermine cholesterylcarbamate (GL-67) disclosed in U.S. Pat. No. 5,767,099. Additionalpreferred lipids include N⁴-spermidine cholestryl carbamate (GL-53) and1-(N⁴-spermidine)-2,3-dilaurylglycerol carbamate (GL-89).

The vectors of the invention may be targeted to specific cells bylinking a targeting molecule to the vector. A targeting molecule is anyagent that is specific for a cell or tissue type of interest, includingfor example, a ligand, antibody, sugar, receptor, or other bindingmolecule.

Invention vectors may be delivered to the target cells in a suitablecomposition, either alone, or complexed, as provided above, comprisingthe vector and a suitably acceptable carrier. The vector may bedelivered to target cells by methods known in the art, for example,intravenous, intramuscular, intranasal, subcutaneous, intubation,lavage, and the like. The vectors may be delivered via in vivo or exvivo applications. In vivo applications involve the directadministration of an adenoviral vector of the invention formulated intoa composition to the cells of an individual. Ex vivo applicationsinvolve the transfer of the adenoviral vector directly to harvestedautologous cells which are maintained in vitro, followed byreadministration of the transduced cells to a recipient.

In a specific embodiment, the vector is transfected intoantigen-presenting cells. Suitable sources of antigen-presenting cells(APCs) include, but are not limited to, whole cells such as dendriticcells or macrophages; purified MHC class I molecule complexed tobeta2-microglobulin and foster antigen-presenting cells. In a specificembodiment, the vectors of the present invention may be introduced intoT cells or B cells using methods known in the art (see, for example,Tsokos and Nepom, 2000, J. Clin. Invest. 106:181-183).

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

Various references are cited herein, the disclosure of which areincorporated by reference in their entireties.

What is claimed is:
 1. A method of identifying variants of SEQ ID NO:7or its complementary sequence comprising (a) isolating genomicpolynucleotide from a subject and (b) determining the presence orabsence of a variant in said genomic nucleic acid using an isolatedpolynucleotide at least 50 nucleotides in length identical to a regionof SEQ ID NO: 7, said region selected from the group consisting of a5′-noncoding region, a 3′-non-coding region and a contiguous coding andnon-coding nucleic acid sequence of SEQ ID NO:7 or reverse strand ofsaid polynucleotide.
 2. The method according to claim 1, wherein said5′-noncoding region consists of nucleotides 7610-17290 of SEQ ID NO:7and said 3′-non-coding region consists of nucleotides 1-7027 of SEQ IDNO:7.
 3. The method according to claim 1, wherein said genomicpolynucleotide is DNA or RNA.
 4. A method of detecting the presence orabsence of a nucleotide variant of SEQ ID NO:7, or its complementarysequence comprising: (a) isolating genomic nucleic acid from a subject,and (b) determining the presence or absence of a nucleotide sequencevariation in said genomic nucleic acid by comparing the nucleotide acidsequence of SEQ ID NO:7 with the nucleotide sequence of the isolatedgenomic nucleic of (a) and establishing if and where a difference occursbetween the two nucleic acid sequences thereby identifying a nucleotidesequence variant of SEQ ID NO:7, or its complementary sequence.
 5. Themethod according to claim 4, wherein said genomic polynucleotide is DNAor RNA.